Recombinant 50S ribosomal protein L15 (rplO)
Description
Molecular Characterization and Structure
The rplO gene encodes a 15 kDa protein that interacts with over ten other ribosomal proteins during 50S subunit assembly in vitro . Structural studies using chemical footprinting and hydroxyl radical probing reveal that L15 binds to nucleotides 572–654 in domain II of 23S rRNA, a region critical for the tertiary folding of the rRNA . This binding requires a partially assembled particle, indicating that L15 stabilizes higher-order structures during ribosome maturation . Mutational analyses further highlight its proximity to rRNA elements in domains I, IV, and V, underscoring its role in coordinating ribosome architecture .
Expression and Purification
Recombinant L15 can be efficiently expressed in E. coli and yeast, offering high yields and shorter production timelines . Expression in insect or mammalian cells is also feasible, enabling post-translational modifications (PTMs) such as glycosylation or phosphorylation, which may enhance protein stability or activity . Under simulated microgravity (SMG), E. coli cultures exhibit higher rplO productivity and plasmid copy numbers, suggesting SMG as a potential optimization strategy for industrial-scale production .
Functional Role in Ribosome Assembly
L15 is indispensable for bacterial viability. Deletion of the rplO gene results in lethality, as demonstrated by chromosomal knockout experiments . Its absence disrupts 50S subunit formation, leading to defective ribosome assembly and impaired translation . Functional studies also reveal that L15 interacts with the peptidyl-transferase center (PTC), the catalytic core of the ribosome responsible for peptide bond formation .
Antibiotic Resistance Studies
Mutations in rplO (e.g., Gly67Asp) have been linked to reduced susceptibility to aminoglycosides like amikacin and gentamicin. These mutations likely alter ribosome conformation, mitigating drug binding to the PTC . Complementation experiments confirm that wild-type rplO restores drug sensitivity, validating its role in resistance mechanisms .
Omics-Based Production Optimization
Transcriptomic and proteomic analyses under SMG reveal upregulation of rplO alongside other ribosome-related genes. This correlates with enhanced protein synthesis and folding efficiency, suggesting that SMG environments can be engineered to maximize recombinant protein yields .
Quantitative Analysis
Selected reaction monitoring (SRM) mass spectrometry has been developed to quantify L15 in E. coli lysates. This method enables precise tracking of nascent and mature r-proteins during ribosome biogenesis, aiding in engineering studies .
